JP5807946B2 - Crystal diffraction measurement method and diffraction measurement apparatus therefor - Google Patents

Description

  The present invention relates to a method for measuring diffraction of crystals, and more particularly to a method for measuring diffraction of hydrous crystals at room temperature and during freezing, and a diffraction measurement apparatus therefor.

  Crystals such as many proteins and some small organic molecules contain about half the volume of water (solvent water) as part of their crystal structure. The crystallinity of these single crystals is impaired. In analytical measurements such as single-crystal X-ray diffraction measurement experiments, a sample (such as a single crystal containing a protein or a small organic molecule) is temporarily stored (mounted) in a fixture that is fixed to the measuring device. )There is a need. In order to prevent drying and the like, various contrivances for suppressing drying are made on the fixture.

The drying control method can be roughly classified into two. One is a sealing method in which the sample is stored in a container, and the other is a freezing method in which a sample is frozen and held at an extremely low temperature of 100K or less (Non-patent Document 1).
In the enclosing method, generally, a crystal and a solvent are enclosed with a glass capillary (capillary) as a fixture to suppress the evaporation of solvent water in the crystal and protect the crystal from drying (Non-patent Document 2). This method is suitable for measurement at room temperature, but is not suitable for a freezing experiment in which a single crystal is rapidly frozen and a diffraction experiment using X-rays or the like is performed. In the freezing experiment, a rapid cooling method by blowing a low temperature gas onto the sample is used, but there is an air layer and a capillary glass layer that are sealed around the crystal, and the heat exchange of the sample is slowed down. It is not possible.

In the freezing method, the crystal sample is scooped together with the surrounding solvent into a polymer ring-shaped fixture (loop) having a diameter equivalent to the size of the crystal without using a capillary, and a rapid gas spray is applied. Freezing is performed (Non-patent Document 3). In recent years, X-ray diffraction experiments are generally performed with high-intensity synchrotron radiation for high-accuracy data measurement. In such cases, radiation damage to samples such as proteins has become a serious problem. The freezing method is useful as a method for suppressing the damage and is widely used.
However, in this freezing method, freezing of solvent water is often a problem. When the solvent water forms hexagonal ice (ice crystal), it causes volume expansion and damages the protein crystal. This phenomenon can occur not only in the solvent water around the sample (single crystal) but also in the same solvent water (crystal water) contained in the sample.

  For this reason, in the freezing method, a reagent called an anti-freezing agent is used in order to suppress the formation of ice crystals generated when the solvent water is frozen. This cryoprotectant is roughly classified into two types. One is an oily anti-freezing agent such as mineral oil or silicone oil, which aims to cover the periphery of the crystal and remove the surrounding solvent water (Non-Patent Document 4). This type of anti-freezing agent not only breaks the crystal by contact with the crystal, but it is also difficult to prevent ice crystal formation derived from crystal water.

  The other is an aqueous anti-freezing agent such as sugar and polyol (glycerol, etc.), which has a hydrophilic group such as a hydroxyl group, and aims to destroy the hydrogen bonding network of solvent water (Non-patent Document 5). Since this type of anti-freezing agent penetrates into the inside of the crystal, the formation of ice crystals can be suppressed also from the inside of the crystal. With the use of such anti-freezing agents, many samples can be processed by the freezing method.

  However, in order to exert the effect of the aqueous cryoprotectant, it is necessary to add the aqueous cryoprotectant to a concentration of several tens of volume percent with respect to the crystal. In general, the interaction of proteins with small amounts (several volume percent) of aqueous cryoprotectants is not strong, but binding to proteins is not negligible at such high concentration conditions, and in some cases, crystal damage is also observed. It is done. For this reason, the type and amount of the aqueous cryoprotectant are the main conditions for the success of the freezing method, and complicated conditions must be studied.

As a solution to this, a method of removing surrounding solvent water as much as possible can be considered. One is a method of preventing drying by blowing an air stream with adjusted humidity on the crystal (Non-Patent Document 6). This humidity adjusting gas spraying method was originally developed as a method for improving the crystallinity (diffraction resolution, etc.) by changing the moisture content of the crystal by adjusting the humidity around the sample and by the structural transition caused thereby. However, it can also be used to maintain crystallinity by combining the wet gas to be blown with the water vapor pressure of the crystal. The sample holding method in this method includes a loop mount method, a crystal resin fitting (mesh loop) with a crystal (MiTeGen, Ithaca, New York, USA, Molecular Dimensions, Newmarket,
England) or a method of holding the crystal by sucking the crystal with a micropipette. However, crystals that are exposed to unadjusted air during the mount operation are often damaged by evaporation of the solvent water in the crystals. It is difficult to remove. Moreover, the more the solvent water is removed, the more difficult it is to fix to the fixture, and stable sample holding is not possible.

On the other hand, a method of sucking crystals with a micropipette and holding the crystals has been developed since then, and a method not based on humidity control has been devised (Patent Document 1). However, since it is difficult to stably hold the sample in a state where the solvent is removed, it is necessary to immediately freeze the sample, and an experiment at room temperature is difficult.
In order to evaluate damage caused by freezing, it is desirable to perform diffraction measurement by X-ray or the like of a sample at both room temperature and low temperature. In addition, although the formation of ice crystals in the surrounding solvent water and the formation of ice crystals in the solvent water in the crystals are generally different, the influence of the solvent water around the hydrous crystals is particularly large, so a method to suppress this effect is required. ing.

JP 2006-53085 A

(Garman, E. F. & Schneider, T. R. (1997). J. Appl. Cryst. 30, 211-237.) (Silfhout, R. G. & Hermes, C. (1995). Rev. Sci. Instrum. 66, 1818-1820.) (Teng, T. Y. (1990). J. Appl. Cryst. 23, 387-391.) (Hope, H. (1988). Acta Cryst. B44, 22-26.) (Petsko, G. A. (1975). J. Mol. Biol. 96, 381-392.) (Reiner, K. et a., (2000). J. Appl. Cryst. 33, 1223-1230., Sanchez-Weatherby, J. et al. (2009). Acta Cryst. D65, 1237-1246.)

  The present invention has been made in view of such problems, and an object of the present invention is to provide a method for preventing the drying of water-containing crystals during diffraction measurement by X-rays or the like and performing appropriate sample freezing, and an apparatus therefor. .

  As a result of intensive studies, the present inventors have conducted a diffraction measurement method including a step of wrapping a crystal with a water-soluble polymer, and an apparatus therefor, in particular, an X-ray diffraction measurement method including a step of wrapping a water-containing crystal with a water-soluble polymer and a device therefor. I found.

In the present invention, an environment (buffer zone) in which the water content of the crystal can be arbitrarily adjusted is provided by wrapping the water-containing crystal such as protein or small organic molecule in a diffraction measurement by X-ray or the like with a water-soluble polymer. When the water-soluble polymer contains an appropriate solvent water, it not only suppresses drying that affects the structure of the crystal, but also freeze-freeze (rapid cooling to 100K or less), so that the crystallinity is not impaired. A frozen sample suitable for analytical measurement such as crystal analysis can be created. Therefore, the concentration of the cryoprotectant for obtaining an appropriate frozen sample can be suppressed to the minimum necessary amount, and in some cases, the cryoprotectant can be omitted.
Further, the water-soluble polymer can be used as an adhesive for fixing the sample and can be taken out in a state where the sample is wrapped from the solvent, so that it can be mounted on the fixture as it is. This can prevent the sample from being directly exposed to air during the diffraction measurement process.

(A) It is a state immediately after mounting a crystal in an air stream after bonding with polyvinyl alcohol (PVAL) which is a kind of water-soluble polymer. (B) A state after the humidity of the mounted crystal of (a) is adjusted. It is an arrangement schematic diagram of low-temperature nitrogen gas and temperature control gas at the time of humidity adjustment. It is an arrangement schematic diagram of low-temperature nitrogen gas and temperature control gas at the time of quick freezing. These are diffraction data after mounting a chicken egg white lysozyme crystal using PVAL 8% having a polymerization degree of 4500 and drying to 79.4% humidity and after holding for 2 hours at around 79.4% humidity. (A) Scattering result after rapid freezing of bovine insulin crystals using only antifreeze (Glycerol 30%). (B) Scattering results after bovine insulin crystals wrapped only with 8% PVAL having a polymerization degree of 4500, dried at 90.4% humidity and rapidly frozen. It is the diffraction data of a chicken egg white lysozyme crystal after drying at 73.9% humidity using PVAL 13% with a polymerization degree of 4500. (A) Diffraction data of bovine insulin crystals after rapid freezing using 20% Glycerol (glycerol). (B) Diffraction data of bovine insulin crystals after quick freezing using Glycerol (glycerol) 30%. (C) Diffraction data of bovine insulin crystals at a humidity of 84.8% using 8% PVAL with a polymerization degree of 4500 and 5% Glycerol (glycerol).

  Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings. The embodiments disclosed individually are examples of the crystal diffraction measurement method and the diffraction measurement apparatus therefor according to the present invention, and the present invention is not limited thereto.

(First embodiment)
The X-ray diffraction measurement method for hydrous crystals according to the first embodiment of the present invention is characterized in that the X-ray diffraction measurement method includes a step of wrapping the hydrous crystals with a water-soluble polymer. That is, the present invention is characterized in that the X-ray diffraction measurement method includes a step of wrapping the water-containing crystal with a water-soluble polymer, but the other essential steps for the X-ray diffraction measurement method are the same as the conventional steps. It is.
The water-soluble polymer herein may be any water-soluble polymer having water retention, and a water-soluble polymer used as a crystal adhesive is particularly preferable. Examples include polyvinyl alcohol (PVAL), carboxymethyl cellulose, polyallylamine hydrochloride polymer, allylamine (free) polymer, allylamine acetate / diallylamine acetate copolymer, polyvinylpyrrolidone and the like. Among these, PVAL is most preferable in the present invention.
PVAL preferably used in the present invention is a polymer having a very strong hydrophilicity and is soluble in hot water. Common uses are office glue, laundry glue, and film. Moreover, since it does not have biotoxicity, it is used also for the method of fix | immobilizing microorganisms and utilizing for fermentation, and the embedding agent for fixing a biological sample by optical microscope observation.
As a result of intensive studies, the present inventors have found that even if the crystal is completely encapsulated in the water-soluble polymer as described later, the water-soluble polymer does not affect the diffraction measurement by X-ray or the like of the crystal. discovered.

The crystal referred to in the present invention refers to a crystal such as a protein or a small organic molecule, and also includes a crystal such as a protein-organic small molecule complex. The water-containing crystal in the present invention refers to a crystal containing a water molecule in the crystal structure, and examples of the water molecule include solvent water when the crystal is formed.
The diffraction measurement method of the present invention is not particularly limited as long as it is suitable for ordinary crystals and particularly suitable for water-containing crystals. The X-ray used for X-ray diffraction may be any X-ray that can be used for a diffraction experiment of the protein crystal or the like. For example, an accelerated electron beam (30 It may be due to the difference in the excitation level of the electrons, or to the acceleration kinetic energy such as suddenly braking the electrons with the counter-cathode or changing the direction of motion by a magnetic field. Moreover, the quantum beam used for the diffraction experiment by the method of the present invention is not limited to X-rays, and neutron beams and electron beams are also used in the same manner.

  “Wrapping the crystal with the water-soluble polymer” included in the method of the present invention means that the crystal is completely wrapped with the water-soluble polymer. A water-soluble polymer having a function as an adhesive is also used when mounting a crystal from the beginning, but when used as an adhesive, it contacts only a part of the crystal, and the mounting tool and the crystal are attached. It is only necessary to make contact, and it is not necessary to envelop the entire crystal. However, since the water-soluble polymer used in the present invention completely encloses the crystal, the water-containing crystal containing water is disconnected from the air by contacting only the water-soluble polymer. Moisture of the water-containing crystal is not in the air but only in dynamic equilibrium with the water-soluble polymer, and the water-soluble polymer constitutes a buffer zone between the water-containing crystal and air.

FIG. 1 (a) shows the crystals (11 and 12) wrapped in PVAL mounted as they are, and FIG. 1 (b) shows the state after the mounted crystals of (a) are humidity-adjusted. The crystals (13 and 14) are still encased in PVAL. As can be seen from this state, in the method of the present invention, in order to prevent the crystal from drying in the X-ray diffraction measurement, the crystal is a water-soluble polymer in the entire process from the removal of the crystal from the solvent to the completion of the X-ray diffraction measurement. You can remain encased. Moreover, the operation is extremely simple.
In addition, the small photograph at the lower right of each photograph in FIGS. 1A and 1B is an image obtained by rotating 90 ° from above.

In the method of the present invention, since there exists an equilibrium state of water between the water-soluble polymer and air and an equilibrium state of water between the water-soluble polymer and the water-containing crystal, the amount of water contained in the water-containing crystal Can be adjusted. For example, when the crystal needs to be in a wet condition, the humidity of the air can be increased and the water content of the crystal can be increased. On the other hand, if you want to lower the water content to freeze the water-containing crystals, if you reduce the humidity of the air, the water in the water-containing crystals will evaporate to the air via the water-soluble polymer and remove the excess water in the water-containing crystals be able to. Thereby, the bad influence to the crystal | crystallization by freezing can be prevented.
In addition, the amount of the cryoprotectant to be introduced in order to prevent the harmful effects of conventional freezing can be minimized. In the method of the present invention, depending on the type and properties of the hydrated crystals, when frozen, the cryoprotectant can be suppressed to 10% by volume or less, or 5% by volume or less. Furthermore, no cryoprotectant may be required.

(Second embodiment)
The second embodiment of the present invention is an X-ray diffraction measurement device including a step of enclosing a water-containing crystal with a water-soluble polymer, including a humidity control device and / or a freezing control device. That is, when the water-containing crystal is wrapped with a water-soluble polymer, in order to adjust the moisture contained in the water-containing crystal through the water-soluble polymer between the water-containing crystal and air, or In order to freeze, it is an embodiment which includes a humidity control device and / or a freezing control device on the outside (the outside portion when the water-containing crystal is wrapped with a water-soluble polymer). The X-ray diffraction measurement device includes a humidity control device, a freezing control device, or a device other than a conventional device that is essential for X-ray diffraction measurement (for example, a diffractometer using a synchrotron radiation or X-ray generation device as a light source) It is characterized by including both.

As the humidity control apparatus of the present invention, any apparatus can be used as long as the equilibrium state of the water can be controlled.
If the ambient temperature is constant, a simple gas humidity control device (22) as shown in FIG. 2 can be used. However, a temperature / humidity control device (not shown in FIG. 2) capable of controlling temperature as well as humidity enables more precise experiments. The freezing control device (21) of the present invention can be any device as long as it can freeze the water-containing crystals, and particularly preferably freezing with a gas (for example, low-temperature nitrogen gas) as shown in FIG. It is a control device.
In the present invention, as shown in FIG. 3, the water-containing crystal (23) fixed to the goniometer (24) can be frozen directly by the gas freezing control device, whereas first, as shown in FIG. It is also possible to adjust the moisture of the hydrated crystals with a control device for temperature and / or humidity with gas, and then freeze with a freezing control device with gas.

  In the X-ray diffraction measurement of the water-containing crystal by freezing, the water contained in the water-containing crystal can be adjusted and frozen as shown in FIGS. Place water-containing crystals such as proteins and small organic molecules bonded with water-soluble polymers in a controlled airflow, change the humidity to evaporate excess water contained in the water-soluble polymer, and make the water-containing crystals suitable water The amount of the crystal can be controlled, and then the crystal can be rapidly frozen to, for example, 100K or less while using a low-temperature gas (nitrogen, helium), liquid nitrogen, or the like while controlling the humidity.

Specific examples of the apparatus for X-ray diffraction measurement of the water-containing crystal of the present invention include the following.
As shown in FIG. 3, the air flow of the humidity control gas is arranged from below, and the air flow of the low temperature blowing nitrogen gas is arranged above. With this arrangement, the flow of low-temperature nitrogen gas is removed from the crystal position when humidity control gas is used. It was possible to quickly freeze by removing the shielding (shutter) of low-temperature nitrogen gas at the same time as the air flow of humidity control gas was retreated.

The humidity control apparatus of the present invention may be a novel humidity controllable airflow spraying apparatus as shown in FIG. 3, and may be a free mount system (Reiner, K. et al. (2000). J. Appl.
Cryst. 33, 1223-1230.) And Humidity-control
Devices such as Device (Sanchez-Weatherby, J. et al. (2009). Acta Cryst. D65, 1237-1246.) May be used. As in the present invention, as a method of quick freezing, an air stream of a low-temperature gas (nitrogen, helium, etc.) is blown to the place where the crystal is placed, and the shield is instantaneously removed by attaching the crystal while shielding the air stream. There is a quick freezing method that freezes, but a method such as filling a container such as a vial with liquid nitrogen and immersing the crystal directly in liquid nitrogen to freeze it (Cipriani, F. et al. (2006). Acta Cryst. D62,
1251-1259) etc. are also mentioned as an example. The humidity control method or rapid freezing method used in these devices can be used in the present invention.

  Examples of the present invention will be described below, and the crystal diffraction measurement method and the diffraction measurement apparatus of the present invention will be described specifically. However, the technical scope of the present invention is not limited to these.

(Method of experiment)
The low temperature blowing gas stream is withdrawn from the sample position, and the nozzle of the humidity control stream is set at the sample position. It is desirable that the tip of the nozzle be as close as possible to reduce the influence of outside air within 10 mm from the sample position. In this experiment, the distance from the sample position to the tip of the nozzle outlet was about 4 mm. The nozzle diameter was 10 mm.

Apply PVAL to a loop or a strip of polyimide film, wrap the crystal out of the crystallization solution, and mount it on the measuring device. PVAL, which is generally used as an adhesive, can adhere and stably hold crystals, so there is no fear of moving or dropping from the measurement position. In addition, it was also effective in the subsequent freezing to embed the crystals in a PVAL solution and to allow the surrounding solvent to be actively absorbed by PVAL.
The humidity adjustment range should be able to be set to a humidity lower than that, with the upper limit being a humidity condition in which X-ray diffraction by hexagonal ice is not observed when the humidity of only a water-soluble polymer is adjusted and quick freezing is performed. In the case of PVAL, the humidity is 92%, which is the lower limit of the humidity at which ice diffraction is observed. However, the humidity conditions enabling rapid freezing also depend on the crystallization conditions, the solvent content of the crystals, and the like.

The work of scooping the crystal from the crystallized solution and mounting it on the measuring device is to expose the crystal for several seconds to several tens of seconds in the air whose humidity is not controlled. In this experiment, the crystal surface is PVAL. Due to the covering, direct contact between the crystal and the atmospheric layer disappeared or decreased. Thus, moisture was taken away in the atmosphere with PVAL and crystals before mounting, but the crystals were not damaged by the presence of PVAL. Sufficiently retained PVAL will dry and concentrate until its saturated water vapor pressure matches the surrounding vapor pressure, while protecting the crystals from drying. In addition, the crystal in this state could keep the crystallinity for about 2 hours under the controlled humidity condition (FIG. 4). As a result, the crystallinity could be confirmed by the X-ray diffraction measurement result while changing the humidity at room temperature.
In addition, when the humidity is maintained at 92% or less and the water content of PVAL is adjusted, X-ray scattering and diffraction derived from PVAL and solvent water give results equivalent to the conventional method using antifreeze (Glycerol 30%). This was confirmed (FIG. 5).

Irradiate X-rays while gradually adjusting the humidity to check the crystal state. The humidity that can be frozen differs depending on the type of crystal, but when the humidity is gradually lowered, crystallographic parameters such as crystal lattice, mosaic width, and diffraction resolution change. This time, this changing point was used as a guide for freezing.
After confirming the change of the crystal, fix the humidity and return to the sample position (the most suitable position to spray the sample) while blocking the low-temperature sprayed gas stream against the sample with the shutter, and retract the nozzle of the humidity control air stream. Immediately after that, the sample is frozen by opening the shutter of the low temperature blowing gas stream.

(Example 1)
・ Example with chicken egg white lysozyme tetragonal crystal Wrap the crystal with PVAL 13% with a polymerization degree of 4500, adjust the humidity to 75%, freeze and measure. Add anti-freezing agent The crystals could be frozen without damaging the crystals (FIG. 6). PVAL 15% polymerization degree 15%, PVAL polymerization degree 2400 PVAL
Similar results were obtained when 18% was used (not shown).

(Example 2)
-Example with bovine insulin When fast frozen with a normal loop mount, the solvent water around the crystal freezes and ice diffraction (25) is observed under the condition that the concentration of the cryoprotectant Glycerol is 20% or less. The crystals were also damaged by freezing (FIG. 7 (a)).
But Glycerol
Crystals immersed in a solution containing 5% anti-freezing agent were converted to PVAL with a polymerization degree of 4500.
After mounting with 8% and adjusting the humidity to 86%, quick freezing and measurement were performed, the crystals could be frozen without damaging the crystals (FIG. 7 (c)). It was confirmed that the result was the same as the result obtained by the conventional method using antifreeze (Glycerol 30%) (FIG. 7B).

The use of PVAL can take away excess moisture around the crystal and control the humidity around the crystal, thereby suppressing the amount of moisture in the entire sample. When rapid freezing was performed in this state, the formation of ice crystals by the solvent water around the crystals was suppressed, and the crystals could be frozen without impairing the crystallinity.
In addition, even when water of crystallization is damaged during freezing, the conventional method requires the addition of an anti-freezing agent that is close to 30% by volume, but in the present invention, the crystal is frozen by addition of 5% by volume. It became possible to do. In this way, by using only the minimum cryoprotectant necessary for freezing the crystal, the effect of the cryoprotectant was reduced and the crystal could be frozen.

  The present invention provides a diffraction measurement method using X-rays and the like and an apparatus therefor that can effectively retain the water content of water-containing crystals at room temperature or during freezing, and can be used particularly for X-ray diffraction measurement of protein crystals. In the structural analysis of protein-drug complexes, it can be expected to suppress the effects of anti-freezing agents that hinder the binding of drugs, and to promote drug development.

11, 12: Crystals wrapped in PVAL 13, 14: Crystals wrapped in PVAL after humidity adjustment 21: Freezing control device 22: Humidity control device 23: Non-measurement crystal 24 (wrapped in PVAL) : Goniometer 25: Ice diffraction

Claims (5)

A diffraction measurement method comprising a step of wrapping a crystal with a water-soluble polymer and a step of measuring diffraction of the crystal in that state.   The diffraction measurement method according to claim 1, wherein the diffraction measurement method is an X-ray diffraction measurement method.   The diffraction measurement method according to claim 1 or 2, wherein the cryoprotectant is 10% by volume or less.   The water-soluble polymer is at least one of PVAL, carboxymethylcellulose, polyallylamine hydrochloride polymer, allylamine (free) polymer, allylamine acetate / diallylamine acetate copolymer, and polyvinylpyrrolidone. The diffraction measurement method according to any one of claims 1 to 3. An X-ray diffraction measurement device, which is used for diffraction measurement including a step of wrapping a crystal with a water-soluble polymer and a step of measuring diffraction of the crystal as it is, and a temperature / humidity control device and / or a freezing control device X-ray diffraction measurement device including